Exposure of human tissue or cells to harmful stimuli such as lipopolysaccharide (LPS), inflammatory mediators, toxic chemicals, and radiation are known to activate immune cells to produce inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, prostaglandin, leucotriene, and nitric oxide (NO). These event causes inflammatory diseases (e.g., arthritis and septic shock), immunological diseases (e.g., graft rejection, autoimmune disease, and diabetes mellitus), and the death of neuron cells.
Under normal condition, NO, an inflammatory mediator is produced in the endothelial cells and macrophages and shows various physiological activities including the induction of cell death, and antibacterial activity. Especially, NO is known to be involved in the maintenance of the homeostasis of blood pressure by the control of relaxation of endothelial cells in the blood vessel. The above-mentioned harmful stimuli (LPS, inflammatory mediators, and radiation exposure) can induce the expression of an inducible nitric oxide synthetase (iNOS), resulting in the over-production of NO. It is the excessive production of NO that induces the above-mentioned diseases. Therefore, an inhibitor of iNOS activity can be a useful therapeutic agent against various inflammatory diseases.
Another inflammatory mediator, prostaglandin, such as PGE2, PGF2a, and PGI2, is a kind of hormone originating from arachidonic acid and involved in various physiological activities. Its production is regulated by cyclooxygenase (COX), a rate-determining enzyme in the production of prostaglandins. Non-steroidal anti-inflammatory and analgesic agent such as aspirin and indometacin, which inhibit the synthesis of prostaglandin by inhibiting COX activity, are known to show an excellent therapeutic effect for arthritis.
TNF-α, which is produced in the macrophages and fibroblasts upon activation by LPS and various inflammatory mediators, induces a cytotoxic effect as well as the production of prostaglandin and other cytokines such as IL-1 and IL-6. TNF-α was originally found as a tumor necrosis factor and was reported to be involved in various diseases, including autoimmune diseases (e.g., rheumatoid arthritis and graft rejection responses), allergic inflammatory diseases (e.g., asthma and atopic dermatitis), and acute immunological disorders (e.g., septic shock and acute liver disease). Therefore, the development of a compound inhibiting TNF-α production is under active investigation. According to recent studies, a mutant mouse having a defective TNF-α receptor is known to have resistance against toxic effects caused by LPS. In addition, a mouse treated with antibodies against TNF-α is known to have an effect on preventing arthritis and toxicity caused by LPS (B. Beutler, Science, 1985, 229, 869; K. Pfeffer et al., Cell, 1993, 73, 457).
Activation of nuclear factor κB (NF-κB) is required for the production of above mentioned NO, prostaglandins, and TNF-α. NF-κB is also known to regulate the expression of a variety of proteins involved in the various cellular responses such as apoptosis, immune responses, and inflammatory reactions.
NF-κB is composed either of homodimer or heterodimer of several proteins including p50 and p65 proteins and exists as inactive forms in the cytoplasm by binding with I-κB protein, the inhibitor of NF-κB. Stimulated by various stimuli such as TNF-α, IL-1, LPS, free radical, and irradiation, however, I-κB kinase (IKK) is activated and thus phosphorylates I-κB. As a result, NF-κB dissociates from I-κB, enters the nucleus, and regulates the expression of various genes involved in the inflammatory or immunological responses (P. A. Baeueric, D. Baltimore, Cell, 1996, 98, 13-20).
In addition NF-κB regulates not only the expression of COX-2, various cytokines including TNF-α, and adhesion molecules such as E-selection, I-CAM, and V-CAM, but also that of apoptosis signals through death receptors such as the TNF-α receptor family (F. H. Epstein, New England Journal of Medicine, 1997, 336(15), 1066-1071; T. Collins, M. A. Read et al., FASEB J., 1995, 9, 899-909). For example, it is known that NK-κB plays an important role in the regulation of the expression of antiapoptotic proteins such as Mn-SOD, zinc finger protein (A20) and inhibitor of apoptosis protein (IAP), which are known to inhibit death receptor-mediated apoptosis process (D. J. Van Antwerp, S. J. Martin et al., Trends in Cell Biology, 1998, 8, 107-111; C.-Y. Wang, M. W. Mayo et al., Science, 1998, 281, 1680-1683). The treatment of a cancer cell with TNF-α or adriamycin induces the activation of NF-κB, which in turn results in the resistant of the cancer cell to TNF-α- or adriamycin-induced cytotoxicity and apoptosis. Therefore, if the activation of NF-κB is inhibited, the cytotoxic effect of anticancer drug increases by the inhibition of expression of antiapoptotic proteins (A. A. Beg, D. Baltimore, Science, 1996, 274, 782-784; C.-Y. Wang, M. W. Mayo, A. S. Baldwin Jr., Science, 1996, 274, 784-789).
Well known anti-inflammatory agents such as glucocorticoid or aspirin inhibit the activation of NF-κB and/or repress the production of NO or prostaglandin as an important anti-inflammatory mechanism of them. Moreover, herbal drugs widely used as an anti-inflammatory agent in oriental folk medicine are known to exert their effects by the inhibition of NF-κB activity (E. Kopp, S. Ghosh, Science, 1994, 265, 956-959; A. Ray, K. E. Prefontaine, Proc. Natl. Acad. Sci. U.S.A., 1994, 91, 752-756). Thus, NF-κB plays not only a central role in the expression of inflammatory mediators in immune cells and neuronal cell death but also an important role in the regulation of the expression of proteins which inhibit the apoptosis of cancer cells induced by TNF-α or anti-cancer agents.
Meanwhile, current status on the development of inhibitors of NO, NF-κB, and TNF-α is described below:
Inhibitors of NO production have focused on the development of agents that specifically inhibit the enzymatic activities of iNOS. For example, analogues of NO precursor such as L-arginine and L-citrulline and derivatives of aminoguanidine and isothiourea are under active investigation (Babu, B. R. B., Griffith O. W., Current Opinion in Chemical Biology, 1998, 2, 491-500).
Curcumin, capsacin, caffeic acid derivative, tetrandrine, and sanguinarine have been reported to inhibit the activation of NF-κB, and dexamethasone, a representative anti-inflammatory agent of glucocorticoid, inhibit NO production by inhibiting the expression of iNOS gene. The molecular mechanism of dexamethasone in the NO synthesis has been known to be the inhibition of NF-κB activation by induction of I-κB, resulting the suppression of iNOS gene transcription (De Vera, M. E. et al., Am. J. Physiol., 1997, 273, 1290-1296).
There are several drugs, which show inhibitory activity for TNF-α production, including IL-4, IL-10 and tumor growth factor-β (TGF-β), and clinical trials using antibody against TNF-α and soluble TNF-α receptor are underway. As synthetic compounds, pentoxifylline, thaliodomide, and SKF 86002, which is a member of bicyclic imidazole, have been developed. Nevertheless, there has yet to be developed any compound to inhibit TNF-α production for the purpose of a therapeutic agent (A. Eigler et al., Immunology Today, 1997, 18, 487-492).
Meanwhile, about 200 diterpene compounds have been reported from Isodon japonicus or close relatives thereof until now. However, there has been no report indicating not only that these compounds inhibit the production of NO, PGE2, or TNF in cell lines stimulated by LPS but also that these compounds inhibit the activation of NF-κB.
Therefore, the present inventors have designed the present invention to show that kamebanin, kamebacetal A, kamebakurin, and exisanin A, each of which is a member of the kaurane diterpene compound isolated from the methanol extract of Isodon japonicus, inhibit not only the activation of NF-κB, but also the production of NO, prostaglandin, and TNF-α in the LPS-stimulated macrophage cells. These four compounds are useful as therapeutic agents for the treatment of inflammatory diseases, immunological diseases, and cancer.